The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are effective therapeutic agents for patients with non-small cell lung cancer (NSCLC) whose tumors harbor somatic activating mutations in EGFR. However, all patients who initially develop dramatic clinical responses will ultimately develop progressive growth of their lung cancer while being treated with gefitinib or erlotinib. Two known mechanisms of acquired (or secondary) resistance have been identified. These include a secondary mutation in EGFR itself (EGFR T790M) occurring in 50% of patients and amplification of the MET oncogene in 20% of patients. Therapeutic strategies including irreversible EGFR inhibitors and the combination of EGFR/MET inhibitors to overcome these secondary resistance mechanisms have been evaluated pre-clinically and are now entering clinical development. However, several fundamental questions regarding the development of resistance remain unanswered. First, do specific gefitinib resistant clones pre-exist and are thus selected for during the course of therapy or do they arise during the course of gefitinib treatment? Second, while second generation kinase inhibitors are likely to have a therapeutic benefit, it is fully anticipated that resistance (tertiary resistance) will also develop to these agents. We have modeled resistance to EGFR kinase inhibitors in vitro using EGFR mutant NSCLC cell lines. Importantly, these gefitinib resistant EGFR mutant NSCLC cell lines develop the same mechanisms of resistance as do tumors from NSCLC patients with EGFR mutations who clinically develop gefitinib or erlotinib resistance and we have used this in vitro model system to identify a previously unsuspected novel mechanism of gefitinib resistance (MET amplification). Thus these cell lines provide a powerful in vitro system in which to study the origins of gefitinib resistance and to determine whether second generation therapies might alter or delay the onset of gefitinib resistance. In addition, they can be used to further determine the mechanisms of tertiary resistance to irreversible EGFR inhibitors and/or to the combination of EGFR/MET inhibitors. Findings from the cell line models will then be used to examine tumor specimens from patients with EGFR mutations who are being treated in clinical trials with gefitinib, erlotinib or second generation kinase inhibitors. These studies will be accomplished through the following specific aims: Aim 1: To determine whether gefitinib resistant clones pre-exist or arise de novo during drug treatment. Aim 2: To determine how therapeutic agents that neutralize known gefitinib resistance mechanisms impact the emergence of drug resistance. Aim 3: To determine mechanisms of tertiary drug resistance in EGFR mutant NSCLC. The pre-clinical studies will likely lead to new treatment paradigms for patients with EGFR mutant NSCLC which will hopefully be associated with an even longer duration of benefit from EGFR targeted therapy than currently achievable. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are effective therapies for subsets of lung cancer patients whose cancers harbor genetic alterations (mutations) in EGFR. Unfortunately, all patients ultimately develop resistance to these drugs. However, second generation therapeutic agents have been developed against specific gefitinib resistance mechanisms. The goal of this proposal is to study drug resistance mechanisms in vitro and using tumors from lung cancer patients with EGFR mutations. PUBLIC HEALTH RELEVANCE: The outlined studies will focus on fundamental mechanisms of gefitinib/erlotinib resistance, on developing the most effective strategy to delay or prevent the emergence of gefitinib/erlotinib resistance and on identifying the mechanisms of resistance to second generation therapeutic agents. The findings from these studies will hopefully lead to treatment approaches that will be associated with an even longer duration of benefit from EGFR targeted therapy than currently achievable. Together findings from this proposal will have major impact in the treatment of EGFR mutant NSCLC.